Journal of Pharmaceutical Analysis

Stability study on an anti-cancer drug 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid (CLEFMA) using a stability-indicating HPLC method

Dhawal Raghuvanshi, Gregory Nkepang, Alamdar Hussain, Hooman Yari, Vibhudutta Awasthi?

2017, 7(1): 1-9.

Abstract(151) PDF(0)

Abstract:
CLEFMA, 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid, is a new chemical entity with anti-cancer and anti-inflammatory activities. Here, we report its stability in solution against stress conditions of exposure to acid/base, light, oxidant, high temperature, and plasma. The identity of the degradation products was ascertained by mass and proton nuclear magnetic resonance spectroscopy. To facilitate this study, we developed and validated a reverse phase high performance liquid chromatography method for detection of CLEFMA and its degradation. The method was linear over a range of 1–100 μg/mL;the accuracy and precision were within acceptable limits;it was stability-indicating as it successfully separated cis-/trans-isomers of CLEFMA as well as its degradation product. The major degradation product was produced from amide hydrolysis at maleic acid functionality caused by an acidic buffer, oxidant (3%hydrogen peroxide), or temperature stress (40–60 °C). The log k-pH profile showed that CLEFMA was most stable at neutral pH. In accelerated stability study we found that the shelf-life (T90%) of CLEFMA at 25 °C and 4 °C was 45 days and 220 days, respectively. Upon exposure to UV-light (365 nm), the normally prevalent trans-CLEFMA attained cis-configuration. This isomerization also involved the maleic acid moiety. CLEFMA was stable in plasma from which it could be e?ciently extracted by an acetonitrile precipitation method. These results indicate that CLEFMA is sensitive to hydrolytic cleavage at its maleic acid moiety, and it is recommended that its samples should be stored under refrigerated and light-free conditions, and under inert environment.

Bioinformatic prediction and functional characterization of human KIAA0100 gene

He Cui, Xi Lan, Shemin Lu, Fujun Zhang, Wanggang Zhang

2017, 7(1): 10-18.

Abstract(105) PDF(0)

Abstract:
Our previous study demonstrated that human KIAA0100 gene is a novel acute monocytic leukemia-associated antigen (MLAA) gene. But the functional characterization of human KIAA0100 gene has remained unknown to date. Here, firstly, bioinformatic prediction of human KIAA0100 gene was carried out using online software;Secondly, human KIAA0100 gene expression was downregulated by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system in U937 cells. Cell proliferation and apoptosis were next evaluated in KIAA0100-knockdown U937 cells. The bioinformatic prediction showed that human KIAA0100 gene was located on 17q11.2, and human KIAA0100 protein was located in the secretory pathway. Besides, human KIAA0100 protein contained a signal peptide, a transmembrane region, three types of secondary structures (alpha helix, extended strand, and random coil) , and four domains from mitochondrial protein 27 (FMP27). The observation on functional characterization of human KIAA0100 gene revealed that its downregulation inhibited cell proliferation, and promoted cell apoptosis in U937 cells. To summarize, these results suggest human KIAA0100 gene possibly comes within mitochondrial genome; moreover, it is a novel anti-apoptotic factor related to carcinogenesis or progression in acute monocytic leukemia, and may be a potential target for immunotherapy against acute monocytic leukemia.

Binding interaction of phosphorus heterocycles with bovine serum albumin:A biochemical study

Swarup Roy, Raj Kumar Nandi, Sintu Ganai, K.C. Majumdar, Tapan K. Das

2017, 7(1): 19-26.

Abstract(67) PDF(1)

Abstract:
Interaction between bovine serum albumin (BSA) and phosphorus heterocycles (PHs) was studied using multi-spectroscopic techniques. The results indicated the high binding a?nity of PHs to BSA as it quenches the intrinsic fluorescence of BSA. The experimental data suggested the fluorescence quenching mechanism between PHs and BSA as a dynamic quenching. From the UV–vis studies, the apparent association constant (Kapp) was found to be 9.25×102, 1.27×104 and 9.01×102 L/mol for the interaction of BSA with PH-1, PH-2 and PH-3, respectively. According to the F?rster's non-radiation energy transfer (FRET) theory, the binding distances between BSA and PHs were calculated. The binding distances (r) of PH-1, PH-2 and PH-3 were found to be 2.86, 3.03, and 5.12 nm, respectively, indicating energy transfer occurs between BSA and PHs. The binding constants of the PHs obtained from the fluorescence quenching data were found to be decreased with increase of temperature. The negative values of the thermodynamic parametersΔH,ΔS andΔG at different temperatures revealed that the binding process is spontaneous;hydrogen bonds and van der Waals interaction were the main force to stabilize the complex. The microenvironment of the protein-binding site was studied by synchronous fluorescence and circular dichroism (CD) techniques and data indicated that the conformation of BSA changed in the presence of PHs. Finally, we studied the BSA-PHs docking using AutoDock and results suggest that PHs is located in the cleft between the domains of BSA.

Fabrication of an electrochemical sensor for determination of doxorubicin in human plasma and its interaction with DNA

Reza Hajian, Zahra Tayebi, Nafiseh Shams

2017, 7(1): 27-33.

Abstract(145) PDF(3)

Abstract:
In this work, an electrochemical sensor was fabricated for determination of an anthracycline, doxorubicin (DOX) as a chemotherapy drug in plasma based on multi-walled carbon nanotubes modified platinum electrode (Pt/MWCNTs). DOX was effectively accumulated on the surface of modified electrode and generated a pair of redox peaks at around 0.522 and 0.647 V (vs. Ag/AgCl) in Britton Robinson (B-R) buffer (pH 4.0, 0.1 M). The electrochemical parameters including pH, type of buffer, accumulation time, amount of modifier and scan rate were optimized. Under the optimized conditions, there was a linear correlation between cathodic peak current and concentration of DOX in the range of 0.05–4.0 μg/mL with the detection limit of 0.002 μg/mL. The number of electron transfers (n) and electron transfer-coe?cient (α) were estimated as 2.0 and 0.25, respectively. The constructed sensor displayed excellent precision, sensitivity, repeatability and selectivity in the determination of DOX in plasma. Moreover, cyclic voltammetry studies of DOX in the presence of DNA showed an intercalation mechanism with binding constant (Kb) of 1.12×105 L/mol.

N-alkylamide profiling of Achillea ptarmica and Achillea millefolium extracts by liquid and gas chromatography-mass spectrometry

Lieselotte Veryser, Lien Taevernier, Evelien Wynendaele, Yannick Verheust, Ann Dumoulin, Bart De Spiegeleer

2017, 7(1): 34-47.

Abstract(106) PDF(2)

Abstract:
Achillea millefolium and Achillea ptarmica are both plants belonging to the Asteracea family and are traditionally used for their medicinal properties. It has already been shown that some N-alkylamides (NAAs) are responsible for these pharmacological actions. Therefore, in the present study, the NAA content of the two plants was analytically characterised. Different extracts were prepared from the roots, the leaves, the stems and the flowers. The structures of NAAs have been assigned in ethanolic extracts of Achillea millefolium and Achillea ptarmica using high performance liquid chromatography – electrospray ionisation – mass spectro-metry (HPLC–ESI–MS) and gas chromatography–electron impact–mass spectrometry (GC–EI–MS). Using both analytical techniques, the structures of 14 and 15 NAAs have been assigned in Achillea ptarmica and Achillea millefolium, respectively. Structures of two new NAAs, previously never observed in Achillea ptarmica, were assigned: deca-2E,6Z,8E-trienoic acid 2-methylbutylamide (homospilanthol) or a related isomeric compound and deca-2E,4E-dienoic acid N-methyl isobutylamide. The structure of homospilanthol or a related isomeric compound was also assigned in Achillea millefolium for the first time.

Analysis of bacitracin and its related substances by liquid chromatography tandem mass spectrometry

Suleiman Ahmed Suleiman, Fan Song, Mengxiang Su, Taijun Hang, Min Song

2017, 7(1): 48-55.

Abstract(146) PDF(4)

Abstract:
A suitable liquid chromatography quadrupole time-of-flight mass spectrometric (LC–Q-TOF–MS) method was developed for separation and characterization of related substances in bacitracin test drug. The separation was performed on LiChrospher RP-18 column using methanol as mobile phase A and 0.2% ammonium acetate buffer solution as mobile phase B in gradient elution. A total of 12 related substances were detected through high resolution mass spectrometric determination in a positive electrospray ionization mode. They were identified as co-existing active components and degradation products of bacitracin through the analysis and elucidation of both the protonated parents and the product ions of all the related substances and their fragmentation pathways were also proposed.

Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography-tandem mass spectrometry method in human plasma

Dhiraj M. Rathod, Keyur R. Patel, Hiren N. Mistri, Arvind G. Jangid, Pranav S. Shrivastav, Mallika Sanyal

2017, 7(1): 56-62.

Abstract(66) PDF(1)

Abstract:
The present study describes a simple, reliable and reproducible liquid chromatography–tandem mass spectro-metry method (LC–MS/MS) for the simultaneous determination of allopurinol and its active metabolite, oxypurinol in human plasma for a pharmacokinetic/bioequivalence study. After protein precipitation (PPT) of 100 μL plasma sample with 1.0%formic acid in acetonitrile, the recovery of the analytes and allopurinol-d2 as an internal standard ranged from 85.36% to 91.20%. The analytes were separated on Hypersil Gold (150 mm×4.6 mm, 5 μm) column using 0.1% formic acid-acetonitrile (98:2, v/v) as the mobile phase. Quantification was done using electrospray ionization in the positive mode. The calibration concentration range was established from 60.0 to 6000 ng/mL for allopurinol and 80.0–8000 ng/mL for oxypurinol. Matrix effect in human plasma, expressed as IS-normalized matrix factors ranged from 1.003 to 1.030 for both the analytes. The developed method was found suitable for a clinical study with 300 mg allopurinol tablet formulation in healthy subjects.

Determination of fenticonazole in human plasma by HPLC-MS/MS and its application to pharmacokinetic studies

Weixing Mao, Yiya Wang, Wenhui Hu, Feifei Jiao, Hongwei Fan, Li Ding

2017, 7(1): 63-70.

Abstract(106) PDF(0)

Abstract:
Two simple and sensitive high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) methods were developed and validated for the determination of fenticonazole in human plasma after percutaneous and intravaginal administration. Mifepristone was used as an internal standard (IS), and simple protein precipitation by acetonitrile containing 2%acetic acid was utilized for extracting the analytes from the plasma samples. Chromatographic separation was performed on a Kinetex XB-C18 column. The quantitation was performed by a mass spectrometer equipped with an electrospray ionization source in multiple reactions monitoring (MRM) positive ion mode using precursor-to-product ion transitions of m/z 455.2–199.1 for fenticonazole and m/z 430.2–372.3 for mifepristone. The validated linear ranges of fenticonazole were 5–1000 pg/mL and 0.1–20 ng/mL in plasma for the methods A and B, respectively. For the two methods, the accuracy data ranged from 85% to 115%, the intra- and inter-batch precision data were less than 15%, the recovery data were more than 90%, and no matrix interference was observed. The methods A and B were successfully validated and applied to the pharmacokinetic studies of fenticonazole gel in Chinese healthy volunteers after percutaneous and intravaginal administration, respectively.

Bile salt induced solubilization of methylene blue:Study on methylene blue fluorescence properties and molecular mechanics calculation

Susithra Selvam, Ivy Sarkar

2017, 7(1): 71-75.

Abstract(94) PDF(1)

Abstract:
Methylene blue (MB) is a hydrophobic drug molecule, having importance both as a staining reagent and pharmaceutical agent. MB is strongly fluorescent, with an emission peak at 686 nm (λex 665 nm). In the study, the possibility of MB as an extrinsic fluorophore to study the micellization behavior of bile salts (BSs) was carried out. Since BSs are drug delivery systems, the solubilization of hydrophobic MB drug molecule by BSs was achieved and the nature of association of MB with BS media, namely sodium cholate (NaC) and sodium deoxycholate (NaDC) was evaluated. Change in the photophysical properties of MB is monitored through fluorescence intensity and fluorescence anisotropy at emission peak, 686 nm of MB. Molecular mechanics calculations were carried out to evaluate the MB–BS association. The estimated heat of formation,ΔHf values are–625.19 kcal/mol for MB–NaC and–757.48 kcal/mol for MB–NaDC. The photophysical study also revealed that MB reports the step-wise aggregation pattern of BSs media, as an extrinsic fluorescence probe.

2017 Vol. 7, No. 1